Skip to main content

Molecular characterization of vesicle tethering complexes as novel regulators of collagen trafficking

Periodic Reporting for period 1 - COTETHERS (Molecular characterization of vesicle tethering complexes as novel regulators of collagen trafficking)

Reporting period: 2017-04-01 to 2019-03-31

Correct maturation of collagen molecules is critical for cellular development and physiology, and for tissue organization into functional compartments, including organs, extracellular matrix and connective tissue. In 2016, a novel pathway regulating collagen trafficking was proposed, and its essential components were identified (Figure 1). Central to this pathway are two trafficking proteins VPS33B and VIPAR (core CHEVI complex) which have been identified as primarily responsible for the trafficking of at least one enzyme responsible of collagen post-translational modifications, the lysyl- hydroxylase 3 (LH3). Genetic mutations in both the core CHEVI complex and its cargo enzyme induce rare but highly invalidating and often lethal phenotypes, characterized by abnormal collagen distribution and/or maturation often linked to impairment in endosomal trafficking. Moreover, the cargo enzyme and its human homologues, LH1 and LH2, have been found implicated in the progression of cancer metastatization. Mechanisms of LH secretion in metastatic cancers have not been identified, although we can speculate about an involvement of the CHEVI complex.
In the time of this Marie Curie Individual Fellowship, I characterized the molecular players constituting the CHEVI complex and their cargo (Figure 1). I obtained structural information of the core CHEVI complex produced in mammalian cell lines, by combining small angle X-ray scattering analysis with cross-linking and native mass spectrometry (MS). In parallel, I contributed to the structural and functional characterization of its cargo LH3, combining X-ray crystallography, electron microscopy (EM), mutagenesis scanning and activity assays and expanded the study to the entire LH family. Overall, the discoveries made in the timefame of COTETHERS project represent to date an invaluable resource for the society, since they will have direct impact on treatment of highly invalidating health conditions.
Overall and in line with what described in the initial project proposal, not only I have made considerable progress towards attaining the structure of the CHEVI complexes, but also I implemented in the host laboratory protocols and technologies of structural biology and contributed to put in place international collaborations.
I could develop efficient strategies to elucidate the fine details of how VPS33B and VIPAR interact with each other, while characterizing additional protein interactors in the extended complexes (i.e. LH3).
I invested time in optimizing the preparation of the CHEVI complex for cross-linking and native MS as well as negative stain for cryo-EM), which require lower sample amount compared to X-ray crystallography and, when combined together, can lead to structures with comparable resolutions. MS data obtained in collaboration with scientists from Martin-Luther-University Halle-Wittenberg - Halle, Germany - started to shed light on the oligomeric assembly of VPS33B-VIPAR and their organization in the three-dimensional space.
In parallel, I conducted functional studies to dissect the two enzymatic activities of LH3, based on the recently published structure (Scietti et al., 2018 and Figure 1c): I eventually obtained structures of LH3 in complex with a series of compounds and screened for their role as potential LH3 inhibitors for the development of targeted therapeutics. Part of the work is now being summarised in two major first-author publications. Information collected on LH3 were crucial to start expanding the study to the entire family of LH enzymes. In this respect, the laboratory could obtain funding through the iNEXT European initiative (PID: 5157), which allowed me to visit top notch European facilities (including NeCEN in the Netherlands) for the analysis of LH samples using cryo-EM. The data collected will constitute milestones to expand the investigations on this family of enzymes and serve as reliable benchmark for the new light-and-electron microscopy facility that is being installed at the University of Pavia.
The impact of the work described was recognised internationally, as proven by participation and invitation to international conferences and workshops. In 2017, I have presented a poster at the EMBL conference “Revolution in structural biology: celebrating the 100th anniversary of Sir John Kendrew”. In 2018 I was selected among the 12 participants of the “Cryo-EM 2018: practical course on cryo-EM sample preparation” at ESRF in Grenoble. The whole training and related cryo-EM experiments were performed using the samples I provided, therefore allowing me to gain hands on experience in sample preparation for cryo-EM experiments while setting up a starting point from where samples conditions could be optimised.
The scientific approach adopted in COTETHERS opened up new ways to look at collagen homeostasis disorders. The generated molecular findings represent a unique resource to start developing targeted therapeutics to prevent cancer metastatization or to treat genetic diseases involving LH enzyme and/or the CHEVI complex. Moreover, recombinant LH enzymes might be used to manipulate collagen molecules in vitro for generation of innovative biomaterials for tissue engineering. In line with the expected outcome of the two ways transfer of knowledge between myself and the host laboratory described in the initial proposal, I could sharpen my profile as biochemist and structural biologist while implementing in the host laboratory large scale set up of molecular biology and biochemistry and innovative technologies of cryo-EM not described in the initial proposal that became crucial for the project success and the progression of my career. Indeed I will be working as a postdoctoral fellow in the field of protein trafficking in one of the top notch structural biology laboratory in Europe, starting from May 2019.
Moreover, I actively contributed to science popularisation events to promote the tools used to make the discoveries described here, with the support of European money. I have been active in high-school training programs at the University of Pavia and participated in the “European Researcher’s night” as a member of the Forneris lab and active part of the EU corner activities (part of the EU project “SHARPER”). As part of the Forneris lab, we had microscopes to show lysozyme crystals to kids and adults, along with protein structures printed using 3D printers. I contributed to the preparation of press releases to announce project developments and publications of relevant results and held a seminar at the University of Pavia to explain to students and young postdocs how to prepare a winning MSCA proposal (Marie-Skłodowska Curie actions: info day” 27 October 2017 | Pavia, Italy). To further increase public awareness, in September 2018 I presented my work in a public event in my hometown Terlizzi, where there are a few cases of collagen related disorders. The event was successful and the community was sincerely happy for being informed of the way public resources are used.
Gender gender balance has been a topic of interest not only for myself but also for the entire host lab and the structural biology hub at the University of Pavia. In May 2018 I joined a group of undergraduate and PhD students in the organization of a seminar with the aim to invite successful women with interesting careers in science and hosted Nancy Grey, the CEO of the presigious GRC conference program.
Molecular players involved in collagen homeostasis